There is a constant need for improved labelling techniques which would allow one to specifically label a protein of interest in order to isolate and/or track such protein of interest under in vitro or in vivo conditions. One particular method is disclosed in WO 02/083937 describing a method for detecting and/or manipulating a protein of interest wherein the protein is fused to O6-alkylguanine-DNA alkyltransferase (AGT) and the AGT fusion protein contacted with a specific AGT substrate carrying a label, whereby the label is transferred to the fusion protein. The AGT fusion protein is then detected and optionally further manipulated using the label. Several mutants of wild type AGT were shown to be better suitable than wild type AGT (WO 2004/031404; Juillerat, A. et al., Chem. Biol. 10:313-317, 2003; Gronemeyer, T. et al., Protein Eng. Des. Sel. 19:309-316, 2006) in such a labelling method, and a wide range of substituted benzylguanines and related heteroarylmethylguanine compounds were described for use in transferring a label to the fusion proteins comprising AGT and AGT mutants (WO 2004/031405).
Simple O2-benzyl-cytosines are known. Freccero, M. et al., J. Am. Chem. Soc. 125:3544-3553, 2003, obtained O2-o-hydroxybenzyl cytosine on reaction of cytosine with o-quinone methide. Ward, A. D. and Baker, B. R., J. Med. Chem. 20:88-92, 1977, describe O2-benzyl cytosine obtained from 2-chloro-4-aminopyridmidine and the sodium salt of benzyl alcohol.